Advances in the technology of oligodeoxyribonucleotide (ODN) synthesis and purification have led to opportunities for de novo gene synthesis. There are two major approaches to the construction of a synthetic gene. The first approach involves the synthesis of ODNs comprising the entire sequence. ODNs are annealed in a piecemeal fashion followed by joining with DNA ligasei. The DNA fragments are then cloned into a plasmid vector either directly or after amplification by PCR. The second approach is a PCR-based methodii in which multiple ODNs belonging to the two strands of the gene sequence concerned are annealed by short overlaps and then extended by a thermal-stable polymerase using overlapped regions as primers. Synthetic genes sequences can be optimized for maximal expression by eliminating rare codons and utilizing optimal codons for a particular species or for multiple host systems, sequence optimization of transcription and translation initiation/termination region, modification of messenger stability, minimizing secondary structure and adjusting GC content. Existing restriction sites can be removed and/or new restriction sites can be added to prepare the gene for future cloning strategies.
Another example of a method for gene assembly involves solid-phase technology, as described by Stahl et al., in “Solid Phase Gene Assembly of Constructs Derived from the Plasmodium falciparum Malaria Blood-Stage Antigen Ag332”iii The oligonucleotide is synthesized while attached to a solid support. The synthetic procedure uses ligases rather than polymerase, in order to link preformed DNA segments. A similar method was described by Hostomsky et al., in “Solid Phase Assembly of Cow Colostrum Trypsin Inhibitor Gene”iv. The authors noted that the yields of full-length oligonucleotides decrease substantially as the length of the oligonucleotide (synthesized gene) increases. This result would be expected for any oligonucleotide/gene assembly process which uses ligase, because there is no proofreading function. Furthermore, there is no possibility to increase the yield by allowing additional cycles of ligation to be performed, such that yields of correctly formed oligonucleotides of the proper length would be expected to drop sharply as the length of the desired oligonucleotide increased.
U.S. Pat. No. 6,386,861 also describes a method for creating a library of DNA oligonucleotides, by recombining or “shuffling” families of oligonucleotides. However, rather than creating the library according to a set of mathematically defined principles, the library is only created according to homology with a previously defined sequence or group of sequences, thereby limiting the scope of the libraries which may be created by using this method.